Proper control of gene expression is essential for organismal development, cellular response to environmental signals, and the prevention of disease states. Transcription is the first step in gene expression and thus is highly regulated. Transcription in all cells is performed by multi-subunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans. Our lab utilizes a range of approaches including molecular biology, genetics, biochemistry and high-throughput sequencing to obtain a detailed understanding of the mechanism and regulation of transcription. To facilitate our studies, we use bacterial RNAP as a model for understanding gene expression paradigms in all organisms.
Transcriptomes are dynamic and responsive to alterations in environmental conditions or growth state. According to the classical model, transcription is regulated primarily through the action of DNA-binding proteins that activate or repress transcription initiation, with a few long-studied exceptions. However, it is now abundantly apparent that cells employ a highly diverse range of mechanisms to control gene expression during all three phases of transcription: initiation, elongation and termination. An overarching goal of our studies is to understand the diversity of regulatory mechanisms that link changes to cellular state to changes in RNAP activity.
- Mechanism and impact of gene expression control by "nanoRNAs"
It had been widely accepted that, in living cells, the initiation of RNA synthesis by RNAP occurs solely via use of nucleoside triphosphate (NTP) substrates, "de novo initiation." Our studies have challenged this conventional paradigm by establishing that under certain cellular conditions a significant fraction of transcription initiation does not occur de novo, but rather relies upon use of 2- to ~4-nt RNAs, "nanoRNAs," that serve as primers for RNAP. Furthermore, we have established that the impact of nanoRNA-mediated priming on gene expression and cell physiology in E. coli is highly significant. Nevertheless, having only recently discovered that nanoRNA-mediated priming occurs in vivo, the full extent to which nanoRNA-mediated priming impacts gene expression and cell physiology across diverse organisms remains a major area of interest and represents a frontier of our current knowledge.
- ‘Epitranscriptomic’ modifications of RNA 5’ ends: NADand CoA-capping
The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization, and translation efficiency and has been proposed to provide a layer of 'epitranscriptomic' gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate "cap" in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. We have shown instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non‑canonical initiating nucleotides (NCINs) for de novo transcription initiation by bacterial RNA polymerase (RNAP). In addition, we have identified key promoter sequence determinants for NCIN-mediated initiation, shown that NCIN-mediated initiation occurs in vivo, and shown that NCIN-mediated initiation has functional consequences by increasing RNA stability in vivo. We have further shown that NCIN-mediated initiation can occur with eukaryotic RNAP II, suggesting that NCIN-mediated "ab initio capping" may occur in all organisms.
Together with our work on nanoRNA-mediated priming, our studies of NCIN-mediated initiation add to an emerging picture that NTPs are not the only substrates for transcription initiation in vivo. In current work, we are determining the full extent to which NCIN-mediated initiation impacts gene expression in bacterial cells and investigating the possibility that NCIN-mediated initiation provides a direct regulatory connection between metabolism and gene expression.
- Development and application of high-throughput sequencing-based approaches for analysis of transcription
During each phase of transcription, RNAP makes extensive interactions with nucleic acids and is responsive to sequence context. In addition, as each phase of transcription is a multi-step process, different steps during initiation, elongation, and termination can be rate limiting for different transcripts, and thereby serve as potential targets for regulation. Thus, predicting how a given transcription unit (i.e. promoter and transcribed region) will respond to alterations in conditions, and identifying the sequence determinants that dictate the response, represents an immense challenge. While structural studies have revealed some RNAP-nucleic acid interactions that modulate transcription, a full understanding of the relationship between nucleic acid sequence and functional output remains a fundamental gap in our knowledge. Thus, my lab seeks to leverage the capabilities of high-throughput sequencing technologies to address this knowledge gap. In this regard, we have developed experimental platforms for massively multiplexed transcriptomics, massively multiplexed protein-DNA crosslinking, and massively multiplexed DNA footprinting (termed "MASTER," "MASTER-XL," and "MASTER-FP," where "MASTER" denotes massively systematic transcript end readout, "XL" denotes crosslinking, and "FP" denotes footprinting).
In published work, we have used MASTER and MASTER-XL to define the sequence determinants and mechanism of transcription start site selection for E. coli RNAP. In current work, we are using MASTER, MASTER-XL, and MASTER-FP to analyze transcription elongation and termination for bacterial RNAP and to define the sequence determinants and mechanisms of transcription start site selection in eukaryotes. In principle, these approaches can be readily adapted to perform a comprehensive mechanistic dissection of any process involving nucleic acid interactions. Thus, although our current studies are focused on transcription, the technical innovations derived from our studies are likely to have wide-ranging applications across many areas of biology.
